The experiments outlined in this proposal are directed at the conformations and dynamics of regions in the energy landscape other than the unique native ground state. In addition to the native conformation, proteins sample partially- and globally-unfolded conformations whose populations and lifetimes are dictated by a Boltzman distribution and the kinetic barriers between them. These non-native states play a determining role in many cellular processes as well as in the folding process itself. Our experimental studies on the energy landscape of RNase H sets the stage for determining how different features in the landscape are encoded in the amino acid sequence. Specifically, the aims of this proposal are: Specific aim 1: Explore the energy landscape of proteins from extremophiles: a. Determine if residual structure in thermophilic RNase H is a cause of the anomalously low ACp and if this is a general mechanism employed by thermophilic proteins. b. Determine and compare the free energy landscape of a psychrophile Specific aim 2: Examine perturbations of the free energy landscape. a. Set up and utilize a generic and robust system to monitor force-induced unfolding at the single molecule level using optical tweezers. b. Probe the energy landscape of RNase H using mutagenesis and misincorporation proton alkyl exchange Specific Aim 3: Uncover sequence determinants for modularity and cooperativity a. Mine the protein database for designable and autonomous regions within proteins b. Develop a poteome-wide assay for rigidity based on protease sensitivity.